Exposure to ionizing radiation from environmental sources is universal. In addition, most cancer patients receive radiation therapy. Ionizing radiation kills cells, in part, by inducing DNA double-strand breaks (DSBs). The DNA-dependent protein kinase (DNA-PK) is crucial to regulation of DSB repair. DNA-PK, which is activated by binding to broken DNA ends, phosphorylates itself, other repair proteins, and signaling molecules such as p53. The hypothesis to be tested is that DNA-PK has a decision-making, or "checkpoint" function. It is proposed that DNA-PK binds initially to DNA ends to form an arrested complex. Synapsis of one DNA end with a compatible, opposing, DNA end leads to kinase activation and checkpoint release, probably via DNA-PK catalytic subunit autophosphorylation. It is proposed that DNA-PK also senses when a DSB cannot be repaired without further processing and responds by recruiting, and potentially phosphorylating, enzymes that are required to process the ends for ligation. Alternatively, if the complex remains unrepairable, DNA-PK may phosphorylate negative regulatory sites in the complex or in tumor suppressor p53. Three specific aims are: (1) To characterize the initial complex formed when DNA-PK binds to an isolated DNA end. Points of DNA-protein crosslinking and the pattern of accessibility of protein and DNA to chemical and enzymatic probes will be characterized. (2) To characterize mechanisms involved in checkpoint release. Changes in protein-DNA contacts and protein conformation in the synaptic complex, relative to the initial complex, will be identified. Sites of DNA-PKcs phosphorylation in the synaptic complex will be identified and their function investigated. (3) To characterize the DNA-PK complex formed on a DNA end that requires processing prior to repair. Complexes will be formed on an oligonucleotide that has a hairpin terminus and is thus unrepairable without nucleolytic processing. The ability of this complex to recruit and phosphorylate the hairpin endonuclease, Artemis, will be characterized. The use of alternative substrates in the absence of Artemis, such as p53, will be investigated. An overall goal is to obtain basic insights into the regulation of DSB repair that will lead to new, mechanism-based strategies for increasing the efficacy of radiation therapy.